The secretory pathway of eukaryotic organisms is of interest since cells can be engineered to secrete a particular protein of interest. The secretory pathway starts by translocation of the protein into the lumen of the endoplasmic reticulum (ER). In the ER the proteins fold into their final three-dimensional conformation and the core part of the N-glycans are attached to them. A quality control mechanism involving the proteins calnexin and calreticulin also resides in the ER, letting only completely folded proteins continue on the secretory pathway to the next compartment (Hammond and Helenius, 1995, Curr. Opinion Cell Biol. 7:523-529). Secretory proteins that do not fold properly are transported back to the cytoplasm by the translocation machinery and are degraded by the proteasome system (Wiertz et al., 1996, Nature 384:432-438).
The folding and glycosylation of the secretory proteins in the ER is assisted by numerous ER-resident proteins. The chaperones like Bip (GRP78), GRP94 or yeast Lhs1p help the secretory protein to fold by binding to exposed hydrophobic regions in the unfolded states and preventing unfavourable interactions (Blond-Elguindi et al., 1993, Cell 75:717-728). The chaperones are also important for the translocation of the proteins through the ER membrane. The foldase proteins like protein disulphide isomerase and its homologs and prolyl-peptidyl cis-trans isomerase assist in formation of disulphide bridges and formation of the right conformation of the peptide chain adjacent to proline residues, respectively. A machinery including many protein components also resides in the ER for the addition of the N-linked core glycans to the secretory protein and for the initial trimming steps of the glycans.
The levels of the chaperone and foldase proteins found in the ER are regulated at the transcriptional level. For each gene there is a basic level of transcription that can be increased in response to various stimuli. A large amount of secretory protein in the ER (secretory load) can induce the mammalian GRP78 gene, and this induction is mediated through the NF-κB transcription factor (Pahl and Baeuerle,1995, EMBO J. 14:2580-2588). Furthermore, the ER chaperone and foldase genes are upregulated when the amount of unfolded protein increases in the ER. This induction has been named unfolded protein response (UPR) and it has been described in mammalian cells, yeast and filamentous fungi (McMillan et al., 1994, Curr. Opinion in Biotechnol. 5:540-545). The induction can be caused by treatment of cells with reducing agents like DTT, by inhibitors of core glycosylation like tunikamycin or by Ca-ionophores that deplete the ER calcium stores. The promoters of mammalian and yeast genes regulated by UPR have a conserved sequence region called UPR element, where the transcription factor responsible for the induction binds.
When the unfolded protein response pathway is active, a signal is tranduced from the ER lumen to the transcription machinery in the nucleus. A protein implicated in the UPR induction is the IRE1 protein of yeast (Cox et al., 1993, Cell 73:1197-1206, Mori et al., 1993, Cell 74:143-156). It is large protein having a transmembrane segment anchoring the protein to the ER membrane. A segment of the IRE1 protein has homology to protein kinases and the C-terminal tail has some homology to RNAses. It is believed that the IRE1 protein may be the first component of the UPR signal transduction pathway, sensing the ER lumen for the presence of unfolded proteins and transmitting the signal eventually to a transcription factor inducing the ER-protein genes. It has been reported that the IRE1 protein oligomerizes and gets phosphorylated when the UPR is activated (Shamu and Walter, 1996, EMBO J. 15:3028-3039). Over-expression of the IRE1 gene in yeast leads to constitutive induction of the UPR (Id.). Phosphorylation of the IRE1 protein occurs at specific serine or threonine residues in the protein.
Another protein reportedly implicated in the regulation of the UPR pathway is PTC2, a yeast protein phosphatase encoded by the PTC2 gene (Welihinda et al., 1998, Mol. Cell. Biol. 18, 1967-1977). The IRE1 protein is phosphorylated when the UPR pathway is turned on (Shamu and Walter, 1996, EMBO J. 15:3928-3039), and PTC2 dephosphorylates the IRE1 protein and regulates the UPR.
It has further been reported that the yeast transcription factor mediating the UPR induction of the chaperone and foldase genes is the HAC1 protein (Cox and Walter, 1996, Cell 87:391-404, Sidrauski et al., 1996, Cell 87:405-413). It belongs to the bZIP family of transcription factors, having a basic DNA-binding region and a leucine zipper dimerisation domain. The binding of the HAC1 protein to the UPR element of ER-protein gene promoters has been demonstrated (Mori et al., 1998, J. Biol. Chem. 273: 9912-9920). The action of the HAC1 protein is regulated by its amount in the cells; none of the protein can be found in uninduced cells and upon UPR induction it appears rapidly. The HAC1 protein amount is dependent of the splicing of the respective mRNA. In uninduced conditions the intron present in the HAC1 gene close to the translation termination codon is not spliced off, and this intron prevents the formation of HAC1 protein by preventing the translation of the mRNA (Chapman and Walter, 1997, Curr. Biol. 7, 850-859, Kawahara et al., 1997, Mol. Biol. Cell 8, 1845-1862). When UPR is induced, the intron is spliced and the mRNA is translated to form HAC1 protein that activates the promoters of its target genes. The HAC1 intron is spliced by an mechanism not currently described for any other system, involving the RNAse activity of the IRE1 protein and a tRNA ligase (Sidrauski and Walter, 1997, Cell 90, 1031-1039, Gonzales et al., 1999, EMBO J. 18, 3119-3132, Sidrauski et al., 1996, Cell 87, 405-413). The unfolded protein response can be induced constitutively in yeast by transformation with a UPR inducing version of the HAC1 gene. (Cox and Walter, supra.)
Thus, as indicated above, there are a number of reports regarding the secretory pathway. Additionally, there are reports on how to increase secretion so as to provide greater yields of heterologous proteins. Greater yields of protein are generally of interest to industry to provide more of a particular protein and to facilitate purification.
For example, in one report random mutagenesis of the host organism has been performed followed by screening for increased yield of a secreted protein. In another report, there has been fusion of a heterologous protein to an efficiently secreted endogenous protein in order to increase the yield of secretion of the heterologous protein. Both of these methods have been of limited success and other methods to improve protein secretion are desirable.
In other studies, there has reportedly been increased yields of secreted heterologous proteins in yeast by either over-expression or deletion of the yeast ER foldase or chaperone genes on an individual or pairwise basis. For example, over-expression of either the protein disulphide isomerase (PDI) or the KAR2 (homologous to the gene for the mammalian ER chaperone BiP) genes in yeast has been shown to increase the extracellular accumulation of certain secreted heterologous proteins (Robinson et al., 1996, Bio/Technology, 12:381-384; Harmsen, et al., 1996, Appl. Microbiol. Biotechnol., 46:365-370). In contrast, deletion of the CNE1 gene, encoding an ER chaperone homologous to mammalian calnexin, reportedly can lead to increased secretion of a heterologous protein (Parlati et al., 1995, J. Biol. Chem. 270:244-253, Harmsen, supra.). The effect of over-expression or deletion of individual or pairs of ER chaperones or foldases has also been reported on in filamentous fungi, however, increased secretion was not obtained. (Punt, et al., 1998, Appl. Microbiol. Biotech, 50:447-454; Wang, et al., 2000, Current Genetics, 37:57-64).
Therefore, it is desirable to provide new methods to increase production of secreted proteins in eukaryotic cells which are simple and consistent. It is also desirable to provide compositions such as novel genes to be used in methods for the increased production of secreted proteins. It is further desirable to provide. eukaryotic cells according to the invention which are transformed with heterologous genes so as to have an increased capacity to produce secreted proteins.